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. Author manuscript; available in PMC: 2010 Jun 1.
Published in final edited form as: Mol Biochem Parasitol. 2010 Mar 23;171(2):89–96. doi: 10.1016/j.molbiopara.2010.03.002

Figure 1. L. mexicana CRK3:CYCA.

Figure 1

(A) SDS PAGE of CRK3his (lane 1) and CYCAhis (lane 2) purified from E. coli and stained with Coomassie blue R-250. (B) CYCAhis binds CRK3. Ni-NTA beads, with (lane 1) or without (lane 2) bound CYCAhis, were incubated with E. coli lysates expressing CRK3, washed, eluted and the eluted protein subjected to Western blot analysis with α-CRK3 antibody. (C) Activation of CRK3:CYCA. Phosphorylation of histone H1 by L. mexicana CRK3:CYCA was performed by mixing increasing quantities of CYCAhis (0 μg-3 μg in 0.5 μg increments from lanes 1-7) to a fixed amount of CRK3his (4 μg, lanes 1-7) in an in vitro kinase assay buffer containing 2.5 μg of histone H1 per reaction and γ-P32-ATP. Lane 8 contains 3 μg CYCAhis only. Phosphorylated histone H1 was detected following SDS-PAGE and autoradiography. (D) SDS PAGE of CRK3T178Ehis purified from E. coli and stained with Coomassie blue R-250. (E) CRK3T178Ehis kinase assay with CYCA. 4 ug of CRK3his (lane 1) or CRK3T178Ehis (lane 2) was incubated with 3 μg of CYCAhis and histone H1 kinase activity assessed as in panel C. H1; histone H1. (F) CRK3T178Ehis kinase assay with CYC6. 3.5 μg of CRK3T178Ehis (lanes 1 and 2) or CRK3his (lane 3) was incubated with (lanes 2 and 3) or without (lane 1) 3 μg of CYC6his and histone H1 kinase activity assessed as in panel C.